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Self-healing dye for single-molecule fluorescence imaging

September 2018.The limited photostability of fluorescent labels is one of the main limitations in single-molecule fluorescence and super-resolution imaging. An attractive strategy to increase the photostability of organic fluorophores relies on coupling them to photostabilizers creating so-called "self-healing" dyes.

A team of scientists from Goethe University Frankfurt and McGill University in Canada report in a new publication the self-healing properties of trisNTA-Alexa647 fluorophores. NTA stands for N-nitrilotriacetic acid and Alexa647 is DNA-tethered single-molecule fluorophore. Although primarily designed to label biomolecules containing an oligohistidine tag, the team hypothesized that the increased effective concentration of Ni(II) triplet state quenchers, a type of photostabilizer, would lead to an improved photostability.

They evaluated photon output, survival time and photon count rate of different Alexa647-labeled trisNTA constructs differing in the length and rigidity of the fluorophore-trisNTA linker. Maximum photon output enhancements of 25-fold versus Alexa647-DNA were recorded for a short tetraproline linker, superseding the solution-based photostabilization by Ni(II).

Steady-state and time-resolved experiments illustrated that the self-healing role of trisNTA is associated with a dynamic excited triplet state quenching by Ni(II). The improvement of photophysical and photochemical properties requires a judicious choice of linker length and rigidity as well as a balance between rapid dynamic triplet excited state quenching versus dynamic/static singlet excited state quenching.